This is an exploratory R21 proposal focused on the development and function of the medial habenula. The habenula is a paired nucleus residing in the dorsal thalamus (epithalamus). It consists of lateral and medial subnuclei which participate in largely distinct neural circuits. Both parts of the habenula send their output fibers to the ventral midbrain via the prominent habenulopeduncular tract or fasciculus retroflexus (FR). The lateral habenula directly innervates the midbrain dopamine and serotonin systems, while the medial habenula sends a prominent cholinergic projection to the interpeduncular nucleus, which in turn innervates the ventral midbrain. In recent work we have shown that the habenula has a pattern of gene expression quite distinct from other forebrain or thalamic areas, and that a homeodomain transcription factor, Brn3a (pou4f1), regulates many of the characteristic molecular features of the medial habenula and a subset of lateral habenula neurons. Little is known about the function of the medial habenula, but its position at the interface between the limbic/basal forebrain and midbrain monoaminergic systems suggests role in reward, attention, learning and drug dependence. For this reason it has been hypothesized that habenular dysfunction may play a role in schizophrenia, and that drug development for nicotine abuse should be targeted to the habenular pathway. Aim 1 will use genetic methods to disrupt the habenulopeduncular pathway by targeted deletion of Brn3a, using expression of Cre recombinase from the Nurr1 (nr4a2) gene locus. We will test the specific hypothesis that the developmental defects in the habenular projections observed in Brn3a knockout mice are due to a cell-autonomous requirement for Brn3a. Aim 2 will use a mixed-genetic/stereotactic injection strategy to specifically ablate the cholinergic component of the medial habenula in adult mice, based on the targeted expression of inducible diphtheria toxin receptor (iDTR). We will test the hypothesis that such lesions will result in a complete loss of cholinergic and glutamatergic innervation of the central IPN by the habenula, while peptidergic innervation of the lateral IPN will be spared. Aim 3 will develop transgenic mice permitting the light inducible, reversible inhibition of habenula activity using targeted expression of the light-activated chloride pump halorhodopsin, fused to a YFP reporter. We will use YFP fluorescence to identify habenula neurons in slice preparations, and test the hypothesis that Brn3a+ neurons exhibit the low-frequency spontaneous depolarizations previously described in the habenula. We will determine whether this activity and induced action potentials can be inhibited by light. Beyond the specific aims of the proposal, these experiments will generate mouse models to be used in future R01 proposals examining the role of complex behaviors related to schizophrenia and nicotine abuse. PUBLIC HEALTH RELEVANCE: The habenula is brain nucleus in the dorsal thalamus of all mammals that connects the forebrain to midbrain centers associated with motivation, reward, attention, and movement. Despite its central location, remarkably little is known about the function of this nucleus. The present proposal will create transgenic mice in which: 1) the habenula is developmentally disconnected from its targets, or 2) genetically defined medial habenula neurons can be selectively ablated by the injection of a toxin, or 3) habenula function can be turned off with light from an implanted fiber optic probe. These mice will be used to address specific questions about how the habenula develops its unique properties, and in future experiments to determine habenula function in memory, learning, attention, and models of schizophrenia and nicotine dependence.